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NEW DEVELOPMENTS IN THE THERAPY OF CHILDHOOD NEPHRO RTIC SYNDROME
Viktória Sümegi, M.D.
PhD Thesis
Supervisor: Prof. Sándor Túri, M.D., Ph.D., D.Sc.
Department of Paediatrics Faculty of Medicine, University of Szeged
Szeged
2010
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PUBLICATIONS IN THE TOPIC OF PhD THESIS
Full papers:
I. Sümegi V, Haszon I, Bereczki C, Papp F, Túri S (2008) Long-term follow-up after cyclophosphamide and cyclosporine-A therapy in steroid-dependent and -resistant nephrotic syndrome. Pediatr Nephrol 23(7):1085-92 (IF2008:2.321)
II. Sümegi V, Haszon I, Iványi B, Bereczki C, Papp F, Túri S (2004) Long-term effects of levamisole treatment in childhood nephrotic syndrome. Pediatr Nephrol 19(12):1354-60 (IF2004:1.440) III . Sümegi V, Kemény É (2006) Atípusos nefrózis szindróma Gyermekgyógyászati Továbbképzı Elıadások Évkönyve, Tiszaparti Esték 2005-2006, 77-81 (IF2006: - ) IV. Sümegi V (2005) Nephrin, podocin, α-aktinin és WT-1 expresszió nefrózis szindrómás gyermekekben Gyermekorvos Továbbképzés 4:61-63 (IF2005: - ) Abstracts: V. Sümegi V, Haszon I, Bereczki Cs, Túri S (2006) Long-term outcome of cyclosporine A and cyclophosphamide treatment in childhood nephrotic syndrome. Pediatr Nephrol 21:1616 (IF2006: - )
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TABLE OF CONTENTS
Publications in the topic of PhD Thesis………………………………………………….. 2
Table of contents…………………………………………………………………………..3
Summary………………………………………………………………………………….. 4
Abbreviations…………………………………………………………………………….. 6
1. Introduction……….………………………………………………………. 7
2. The aims of the study…………………………………………………….. 10
3. Patients and methods ……………………………………………………... 11
3.1 Levamisole study…………………………………………………………. 11
3.2 CP versus CSA study……………………………………………………... 12
3.3 Definitions…………………………………………………………............13
3.4 Corticosteroid protocol……………………………………………............ 14
3.5 Clinical assessment……………………………………………………….. 14
3.6 Statistical analysis…………………………………………………............ 14
4. Results…………………………………………………………………….. 15
4.1 Results of the levamisole study……………………………………………15
4.2 Results of the CP treated group………………………………………....... 22
4.3 Results of the CSA treated group………………………………………… 25
5. Discussion…………………...……………………………………………. 29
5.1 Discussion of the levamisole study……………………………………….. 29
5.2 Discussion of the CP versus CSA study………………………………….. 31
5.2.1 Clinical course of nephrotic syndrome…………………………………….31
5.2.2 Renal function, proteinuria and hypertension…………………………….. 32
5.2.3 Renal histology………………………………………………………….... 32
5.3.4 Adverse events……………………………………………………………. 33
5.2.5 The possible use of cyclophosphamide or cyclosporine-A for treatment.... 33
6. Conclusions…………………………………….…………………………. 37
Acknowledgement………………………………………………………………………... 38
References…………………………………………………………..……………………. 39
Publications………………………………………………………………………………. 47
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SUMMARY
INTRODUCTION: Eighty to ninety percent of children with steroid-sensitive nephrotic
syndrome (SSNS) have relapses. About half relapse frequently and are at risk of the adverse
effects of corticosteroids. Non-corticosteroid immunosuppressive agents are used to prolong
periods of remission; however these agents have significant potential adverse effects.
Currently there is no consensus as to the most appropriate second line agent in steroid–
unresponsive nephrotic syndrome (NS) in children.
OBJECTIVES: Our objective is to evaluate the benefits and harms of some non-
corticosteroid immunosuppressive agents (i.e. levamisole, cyclophosphamide (CP),
cyclosporine A (CSA)) in frequently relapsing (FRNS), steroid-dependent (SDNS) and
steroid-resistant NS (SRNS) in children.
PATIENTS AND METHODS: A retrospective study was made on the medical records of 34
children (21 boys, 13 girls) with FRNS (15/34), SDNS (13/34), or SRNS (6/34), whom
levamisole was introduced (2 mg/kg per day) after 4 weeks of daily corticosteroid treatment.
A second retrospective study was made on the medical records of 37 children, where two
groups of patients were identified. Group 1 consisted of 22 children (15 boys, 7 girls) who
received CP first as a second-line immunosuppressive drug because of their nephrotic
syndrome. Group 2 consisted of 15 children (10 boys, 5 girls) who received second-line
treatment with CSA.
CONCLUSIONS: Our findings suggest that levamisole significantly reduces the relapse rate
and the cumulative steroid dose in children with FRNS and SDNS and is also a beneficial
and safe therapy for SRNS patients. Levamisole may have a place in preventing relapse even
after courses of akylating agents, and it may also have some benefit for the treatment of
SRNS patients. CP and CSA are effective second-line therapies following steroid
monotherapy in idiopathic NS patients, but the relapse rate is lower and the relapse-free
period is significantly longer in our CP-treated group. A good remission rate still can be
achieved after 5 years following the initial CP and CSA therapy, and the incidence of side
effects is low. An important message of our study is that most children who have a difficult
course of nephrotic syndrome after an initial remission induced by steroids do well 7-8 years
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after presentation. On the other hand, our study is finally not able to give a definitive answer
to the question as to which is the better treatment in the examined patient population. This
retrospective study works with non equal distribution of focal segmental glomerulosclerosis
(FSGS) in the different treatment groups so the question can not be answered due to
statistical restrictions. In addition, no accompanying genetic studies were performed, which
means that about 10% of our patients are expected to have a genetic disorder.
Further research is still needed to elucidate the disorder’s molecular pathogenesis,
identify new prognostic indicators, and to develop better approaches to treatment.
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ABBREVIATIONS
BAPN, British Association for Paediatric Nephrology
BP, blood pressure
Chl, chlorambucil
Clcreat, endogenous creatinine clearance (ml/min per 1.73 m2)
CSA, cyclosporine-A
CP, cyclophosphamide
eGFR, estimated glomerular filtration rate
F, female
IgM NP, immunglobulinM nephropathy
IL, interleukin
INS, idiopathic nephrotic syndrome
ISKDC, International Study of Kidney Diseases in Children
FRNS, frequently relapsing nephrotic syndrome
FSGS, focal segmental glomerulosclerosis
MCNS, minimal change nephrotic syndrome
MMF, mycophenolate mofetil
MP, methylprednisolone
M, male
NF-AT, nuclear factor of activated T-cells
NKF K/DOQI CKD, National Kidney Foundation Kidney Disease Quality Outcomes
Initiative stages for chronic kidney disease
NS, nephrotic syndrome
RRT, renal replacement therapy
SD, standard deviation
SDNS, steroid dependent nephrotic syndrome
SRNS, steroid resistant nephrotic syndrome
SSNS, steroid sensitive nephrotic syndrome
UP, urinary protein
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1. INTRODUCTION
Idiopathic nephrotic syndrome (NS) is the most frequent glomerular disease in children and
is mainly due to minimal change nephrotic syndrome (MCNS) and focal segmental
glomerulosclerosis (FSGS) [1−4]. NS generally has a favourable long-term prognosis, about
90 % of affected children exhibit an excellent glucocorticoid responsiveness, but most of
them suffer at least one relapse [5]. Steroid-responsiveness is of greater prognostic use than
renal histology [6].
According to the literature, about 40 % of the steroid-sensitive (SSNS) cases are frequently
relapsing (FRNS) and they commonly become steroid-dependent (SDNS) [7]. Steroid
resistance (SRNS) develops in 10 % of children and many of these exhibit FSGS [8].
Treatment of the these NS patients is still challenging because they often require
long-term, high-dose immunosuppression (i.e. use of steroids), with a greater prevalence of
side effects and complications (poor growth, cushingoid obesity, adrenal suppression,
hypertension, osteoporosis, cataracts, and psychological disturbances) [5, 9−11]. The
therapeutic aims are to induce complete remission, reduce the rate of relapses, the cumulative
dose of corticosteroids, mortality, therapeutic side effects, and the incidence of serious
complications [12]. In the past two decades, alkylating agents such as cyclophosphamide
(CP) and chlorambucil (Chl), the immunomodulatory drug levamisole, calcineurin inhibitors
such as cyclosporine A (CSA) and recently tacrolimus (TAC) and mycophenolate mofetil
(MMF), the inhibitor of purine biosynthesis, have been used as the main steroid-sparing
agents [13−15]. The effectiveness of cytotoxic therapy depends on the histology of the
lesion, they vary in potency and side effects, and there has been no consensus as to which
should be used as the first choice of second-line drug [16− 18].
CP has been used in nephrology since the 1960s, whereas CSA and levamisole were added
to the armamentarium of therapies in 1987, and a consensus guideline for the treatment of NS
was published by the British Association of Paediatric Nephrology (BAPN) in 1994 [19].
Levamisole was originally developed as an antihelminthic drug with a non-specific
immunomodulatory effect. It has been hypothesized to normalize deficient cell-mediated
immunity [10]. It enhances T-cell responses by stimulating T-cell activation and
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proliferation; it potentiates monocyte and macrophage functions, including phagocytosis and
chemotaxis, and increases neutrophil mobility, adherence and chemotaxis [20]. This
mechanism is important, because SSNS has long been thought to be due to lymphocyte-
derived circulating factors leading to podocyte injury with subsequent proteinuria. New
studies support this mechanism and implicate the role of T helper 2 cytokine inteleukin (IL) -
13. In addition a genetic mutation in familial NS has been reported in a child, who responded
to corticosteroid therapy and preliminary trial data on MMF supporting its efficacy as a
steroid-sparing agent. Among the different drugs used for their steroid-sparing effect in
FRNS, levamisole is the least toxic, and the least expensive. However, it is neither approved
for this indication nor widely used in Europe, there are new clinical trial data supporting the
efficacy of levamisole in SSNS [21].
The alkylating agent CP is widely used in SDNS and FRNS in children, either orally (2-3
mg/kg per day for 8-12 weeks) or in intravenous form (500-750 mg/m2 per month for 6
months) [22]. CP has been shown to prevent progressive scarring within the kidney, preserve
renal function, induce remission, and to reduce the risk of end-stage renal failure, but it also
causes lymphopenia, decreases immunoglobulin secretion, suppresses some of the T-cell
functions and enhances the immune response by inhibiting suppressor T-cells.
The toxicity of CP (2-3 mg/kg per day) [23− 25] and chlorambucil (0.2 mg/kg per day for 8-
12 weeks) [26, 27] is generally mild and reversible, but it includes bone marrow depression,
hemorrhagic cystitis, hair loss, infertility seizures, and rarely, oncogenesis [26−28]. In SDNS
and FRNS there is no significant difference between CP and Chl therapeutic results.
CSA, a lipophilic decapeptide, is well recognized to be effective in the treatment of children
with SDNS or SRNS. CSA is thought to bind to the cytosolic protein cyclophilin
(immunophilin) of immunocompetent lymphocytes, especially T-lymphocytes. This complex
of CSA and cyclophilin inhibits calcineurin, which is responsible for activating the
transcription of interleukin-2 in T-cells. Activation of the T-cell receptor normally increases
intracellular calcium, which acts via calmodulin to activate calcineurin. Calcineurin then
dephosphorylates the transcription factor NF-AT (nuclear factor of activated T-cells), which
moves to the nucleus of the T-cell and increases the activity of genes coding for IL-2 and
related cytokines. CSA prevents the dephosphorlyation of NF-AT by binding to cyclophilin.
It also inhibits lymphokine production and IL release and, therefore, leads to a reduced
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function of effector T-cells. Several mechanisms have been postulated to explain the CSA-
induced reduction in proteinuria in NS. The main mechanisms have been: changes in the
properties of the glomerular barrier, resulting in an increased charge and size selectivity [30];
reduction in glomerular plasma flow or ultrafiltration pressure, which reduces proteinuria on
a haemodynamic basis [30]; and inhibits the expression of IL-2 receptor at transcriptional
level [31].
CSA can be used in teenage boys to avoid gonad toxicity of CP or in children with steroid
toxicity. Response rates of 50-100% have been reported in the literature when twice-daily
dosing of 5 to 32 mg/kg per day is applied (the usual dose is 5-6 mg/kg per day), blood levels
of 70-500 ng/ml being achieved [29]. The efficacy and toxicity of CSA correlate with its
serum concentration. Although there is no standard protocol, the initial CSA treatment
normally lasts for 1-2 years [6].
The most common side effects of CSA are nephrotoxicity, a transient increase in serum
creatinine concentration, a decreased glomerular filtration rate, gingival hyperplasia,
hypertrichosis and gastric discomfort [9, 12, 17, 32].
Because of the relatively low prevalence of FRNS, SDNS and SRNS, it is difficult to
establish the precise stage at which a steroid-sparing agent should be prescribed to control the
disease and minimize steroid toxicity.
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2. THE AIMS OF THE STUDY
The aims of our first study were
1. to investigate the effects of levamisole on the number of relapses in FRNS, SDNS,
and SRNS patients in childhood.
2. to investigate the effects of levamisole on the cumulative steroid dose in FRNS,
SDNS, and SRNS paediatric patients.
3. to investigate the side effects of the levamisole treatment.
The aim of the second study was
4. to compare the effect and
5. to compare the long-term outcome of CP and CSA therapy in those idiopathic NS
(INS) patients who were originally steroid sensitive but after several relapses became SRNS
or SDNS.
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3. PATIENTS AND METHODS
3.1. Levamisole study
A retrospective study was made on the medical records of 34 children (21 boys, 13
girls) with FRNS (15/34), SDNS (13/34), or SRNS (6/34) who were admitted to our
Department of Paediatrics between January 1998 and January 2003. Their ages at the time of
diagnosis ranged between 1.5 and 15 years (median 4.5 years).
Inclusion criteria were age at onset >1 year and <16 years, initial steroid sensitivity,
FRNS or SDNS. Exclusion criteria were NS secondary to other systemic diseases or
syndromes, and renal histology at onset or subsequently consistent with
membranoproliferative or membranous nephropathy.
The first 34 patients who were admitted to our department for FRNS, SDNS, or
SRNS and were treated with levamisole were included in the study. Renal biopsy was
performed in 23 children and showed MCNS in 13, IgM nephropathy (IgM NP) in 7, and
FSGS in 3. Renal biopsy was not undertaken at the initial presentation in 11 steroid-sensitive
children in the absence of risk factors indicative of histology other than minimal change
disease. However, renal biopsy was performed prior to the introduction of cytotoxic therapy
in children with FRNS or SDNS, or in those who developed steroid resistance. In the SRNS
patients, levamisole was introduced following cytotoxic therapy when a new relapse
developed. Prednisolone was restarted because we wanted to investigate whether these
patients would become steroid sensitive with a combined administration with levamisole.
There were 19 patients that received other immunosuppressive therapy before
levamisole [9 received CP (2-2.5 mg/kg per day for 8-12 weeks), 10 received Chl (0.2 mg/kg
per day for 8 weeks)].
Levamisole was introduced (2 mg/kg per day) after 4 weeks of daily corticosteroid
treatment. Following another 4 weeks of alternate-day prednisolone, the dose of steroid was
gradually tapered by 10 mg/week. The duration of levamisole treatment was 17±7 months
(mean±SD; range: 5-36 months). Levamisole was discontinued for 6 months when
leucopenia (white blood cell count < 3x109/l) occurred and was then restarted.
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The level of proteinuria at the time of diagnosis was 4.13±2.51 g/day (mean±SD).
Before the start of levamisole treatment, the proteinuria level was 2.17±1.34 g/day
(mean±SD), the endogenous creatinine clearance was 108.0±47.7 ml per 1.73 m2 (mean±SD),
and the relapse rate was 4.41/year (mean). The cumulative steroid dose before the
introduction of levamisole was 7,564.4±3,497.1 mg/year (mean±SD).
3.2. CP versus CSA study
A retrospective study was made of the medical records of 37 children (25 boys, 12
girls) with idiopathic NS (INS) who were admitted to the Department of Paediatrics between
1989 and 2000 (follow-up time 5-13 years, median 7.1 years). At the start of their disease, all
were steroid sensitive, but following several relapses, they became steroid dependent or
steroid resistant.
Two groups of patients were identified. Group 1 consisted of 22 children (15 boys, 7
girls) age range 2-14 (mean±SD: 7.4±3.6 years) who received CP first as a second-line
immunosuppressive drug because of their NS. Group 2 consisted of 15 children (10 boys, 5
girls) who received second-line treatment with CSA. Their ages at the time of the diagnosis
lay in the interval 5-16 (mean±SD: 11. 7±4.4) years.
Inclusion criteria were age at onset > 1 year and ≤ 18 years, SDNS or SRNS.
Renal biopsy was performed in all cases, and it was indicated because we wanted to
see the histology before the change of steroid therapy to another immunosuppressant.
Biopsy revealed MCNS in 19 and FSGS in three patients in group 1. In group 2, FSGS was
found in seven and MCNS in eight children.
CP was introduced at 2-2.5 mg/kg per day orally for 8-12 weeks. Mean duration of
CP treatment was 2.5±0.5 months (2-3 months). CP therapy was associated with 1 mg/kg
oral prednisolone every other day.
The mean proteinuria level at the time of diagnosis was 5.0±3.5 g/day (mean±SD), which
became <0.5 g/day during the initial steroid therapy. Before the start of CP treatment,
proteinuria level was 3.9±2.9 g/day (mean±SD), endogenous creatinine clearance was
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100.4±50 ml/min per 1.73 m2 (mean±SD), and the relapse rate was 3.4±2.8/year (mean±SD).
The cumulative steroid dose before CP introduction was 6,941±2,891.2 mg/year (mean±SD).
CSA was introduced at 3-5 mg/kg per day. Mean CSA treatment duration was 28±15
(7-60) months. CSA serum drug level was monitored monthly; target concentration levels
were 100-200 ng/ml at trough and 800-1000 ng/ml at peak. CSA therapy was associated with
1 mg/kg prednisolone every other day.
Proteinuria level at the time of diagnosis was 4.5±2.3 g/day (mean±SD) and became <0.5
g/day at the end of the first steroid course. Before the start of CSA treatment, the mean
proteinuria level was 3.9±2.3 g/day (mean±SD), the endogenous creatinine clearance was
86.6±27.3 ml/min per 1.73 m2 (mean±SD), and the relapse rate was 3.7+3.1/year (mean±SD).
The cumulative steroid dose before CSA introduction was 7,656.4±3,517.2 mg/year
(mean±SD).
3.3. Definitions
FRNS was defined as two or more relapses within the first 6 months of the initial
episode or four or more relapses during any 12-month period.
SDNS was defined as at least two relapses during alternate-day steroid treatment or
within 14 days after stopping steroid therapy.
SRNS was defined as no improvement in proteinuria after 1 month of prednisolone
therapy of 60 mg/m2 per day.
Relapse was defined as proteinuria >1.0 g/day (40 mg/m2) for 3 consecutive days.
Complete remission was defined as a reduction in urinary protein excretion to <0.1
g/day (<4 mg/m2) for 3 consecutive days.
Partial remission was defined as decreased urinary protein by >50 % from the
baseline on initial presentation.
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3.4. Corticosteroid protocol
The initial corticosteroid protocol at the start of the disease was prednisolone 60
mg/m2 daily in two divided doses for 4 weeks, followed by 40 mg/m2 in a single dose every
other day for 4 weeks, with the dose then being tapered by 10 mg/week according to the
recommendations of the International Study of Kidney Disease in Children (ISKDC)
protocol. Relapses were treated by the same method in all patients. All patients were steroid
sensitive at the first course of treatment, but they became FRNS, SDNS or SRNS later on.
They received more than one steroid course; the median number was four.
3.5. Clinical assessment
Patients were checked monthly. At each visit, besides the clinical assessment, the
following investigations were performed in every case: urinanalysis, 24-h urinary protein /
urinary protein:creatinine ratio, endogenous creatinine clearance (based upon 24 hours urine
collection), complete blood count, serum creatinine determination, liver function tests were
measured. In the second study (CP versus CSA study) estimated glomerular filtration rate
(eGFR) was calculated according to the Swartz formula: GFR = k x height[cm]/serum
creatinine[mg/dL] where k=0.55, serum lipid profile and CSA blood concentration
determinations were also performed. Genetic analysis was not performed.
At the start of the study, all the patients had normal liver function tests and a normal blood
count in the levamisole group. The endogenous creatinine clearance was initially abnormal in
7/22 patients in the CP and in 5/15 patients in the CSA group.
3.6. Statistical analysis
The clinical data on the patients are reported as median, mean ± standard deviations
(SD). Statistical analyses included the Student’s t-tests for the comparison of parametric data.
The level of statistical significance was taken as ∗p<0.05.
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4. RESULTS
4.1. Results of the levamisole study
Of the 34 patients, 28 had SSNS and 13 of these became steroid dependent. Of the 34,
6 developed SRNS before starting levamisole treatment. The patients showed signs of steroid
toxicity, including growth retardation, obesity, hypertension, or osteoporosis. Data on
patients in the FRNS, SDNS, and SRNS groups are shown in Tables 1, 2, and 3, respectively.
The duration of levamisole treatment was 5-36 (median 17) months. There were 29
patients that received levamisole for at least 18 months, 5 received it for a shorter period
because of leucopenia. Of the 29 children, 23 received levamisole for 18 months; 6 had
longer therapy because of relapses. The level of proteinuria was 4.13±2.51 g/day at the time
of diagnosis and 2.17±1.34 g/day before the start of levamisole treatment.
During therapy the level of proteinuria fell significantly to 0.128±0.213 g/day
(p<0.0001) and remained low after the cessation of the levamisole adjuvant therapy
(0.134±0.301 g/day).
The relapse rate was 4.41/year before levamisole treatment and 0.41/year during
levamisole therapy (p<0.0001). No relapse occurred in 23 of the 34 patients during this
therapy, while 10 children had one, and only one child had two relapses per year. Following
the levamisole treatment the relapse rate during the 24-month follow-up was 0.22/year; 28 of
the 34 children remained in complete remission and only six of them relapsed. The changes
in the level of proteinuria and the cumulative steroid dose are shown in Table 4.
In 23 of the 34 children, steroid administration could be stopped and they were in
remission on levamisole alone. Of the 34, 11 still needed prednisolone treatment because of
relapses and in one the treatment was supplemented with CSA.
There were 2 of 15 of the FRNS patients, 6 of 13 of the SDNS patients, and 3 of 6 of
the SRNS patients that suffered a relapse during the levamisole therapy. The corresponding
rates were 3 of 15 in the FRNS group, 2 of 13 in the SDNS group, and 1 of 6 in the SRNS
group two years after cessation of levamisole therapy (Table 5).
The connections between the histopathology and the therapeutic response during the
treatment and the follow-up are shown in Table 6.
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Table 1 Patients with frequently relapsing nephrotic syndrome (FRNS)
Proteinuria (g/day) Cumulative steroid dose (mg) Patient no.
Sex Age (years)
Biopsy Immunosuppression other than prednisolone before levamisole treatment
At diagnosis At start of After levamisole levamisole therapy therapy
Before start of During levamisole levamisole therapy therapy
1 M 5 MCNS CP 2.3 0.98 0 9240 0 2 M 3 - - 2.3 1.8 0.54 4930 0 3 M 2 - Chl 4.1 2.7 0 1850 0 4 F 4.5 MCNS - 3.2 2.7 0.01 7390 3080 5 F 3.5 MCNS - 2.1 1.1 0 11080 0 6 M 3 - CP 4.8 2.4 0.612 7390 0 7 F 6 IgM NP CP 1.5 1.8 0.1 11090 0 8 F 4 - - 2.1 0.98 0 8320 3080 9 F 5 - CP 5.0 1.5 0.17 3080 0 10 M 1.8 - - 1.5 0.9 0 3400 1680 11 F 2.5 - - 2.1 0.7 0 15400 0 12 F 5 MCNS Chl 8.7 3.5 0 12320 0 13 M 4.5 MCNS Chl 3.7 2.3 0 7530 4920 14 M 4.5 - - 1.7 0.98 0.05 6460 2060 15 F 5 - - 1.4 1.1 0.016 6080 4920
M male; F female; MCNS minimal change nephrotic syndrome; IgM NP IgM nephropathy; CP cyclophosphamide; Chl Chlorambucil.
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Table 2 Patients with steroid-dependent nephrotic syndrome (SDNS)
Proteinuria (g/day) Cumulative steroid dose (mg) Patient no.
Sex Age (years)
Biopsy Immunosuppression other than prednisolone before levamisole treatment
At diagnosis At start of After levamisole levamisole therapy therapy
Before start of During levamisole levamisole therapy therapy
1 M 6 MCNS MP, CP 6.0 2.5 0.54 12320 0 2 M 5 - - 2.9 1.2 0.1 9240 3080 3 M 15 FSGS - 5.2 2.4 0.36 13860 5470 4 F 7 MCNS Chl 6.0 2.6 0.01 6160 0 5 M 1.5 - - 5.4 3.2 0 3330 1100 6 M 14 MCNS CP 11.16 2.6 0 6160 1680 7 M 4 MCNS Chl 5.1 2.4 0.1 3080 0 8 F 6 MCNS Chl 8.8 6.1 0.02 6160 0 9 M 4 IgM NP MP 2.5 2.5 0.4 6160 3080 10 M 4.5 IgM NP CP 4.0 1.15 0.8 6160 2800 11 M 5 FSGS - 10.3 5.8 0.08 6510 2050 12 M 5 IgM NP MP 3.7 4.4 0.2 8360 2050 13 M 5 MCNS - 4.93 2.4 0 5600 0
M male; F female; MCNS minimal change nephrotic syndrome; MP methylprednisolone; CP cyclophosphamide; FSGS focal segmental glomerulosclerosis; Chl chlorambucil; IgM NP IgM nephropathy.
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Table 3 Patients with steroid-resistant nephrotic syndrome (SRNS) Proteinuria (g/day) Cumulative steroid dose (mg) Patient
no. Sex Age
(years) Biopsya Immunosuppression other
than prednisolone before levamisole treatment
At diagnosis At start of After levamisole levamisole therapy therapy
Before start of During levamisole levamisole therapy therapy
1 F 1.5 MCNS Chl 5 3.3 0.02 2870 0 2 F 1.5 IgM NP CP 2.5 0.4 0.01 6600 0 3 M 2 IgM NP MP, CP 2 0.5 0.024 5540 3900 4 M 15 FSGS Chl 2.5 1.5 0.15 11370 2050 5 M 3 MCNS Chl 2.4 1.8 0.32 14690 3080 6 M 5.5 IgM NP Chl 3.4 1.7 0.18 7390 0
M male; F female; Chl Chlorambucil; MP methylprednisolone; CP cyclophosphamide MCNS minimal change nephrotic syndrome; IgM NP IgM nephropathy; FSGS focal segmental glomerulosclerosis.
a Patients had a history of MCNS (in patient 4 FSGS). MCNS was combined with IgM NP in patients 2, 3, and 6.
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Table 4 The distribution and data of patients with nephrotic syndrome FRNS (n=15) SDNS (n=13) SRNS (n=6) Sex (male/female) 9/6 10/3 6/0 Age at diagnosis (years; mean±SD) 3.95±1.25 6.3±3.86 4.75±5.24 Age at start of levamisole therapy
(years) 6.67±3.18 9.37±4.1 6.2±5.6
Proteinuria (g/day) at diagnosis at start of levamisole therapy after levamisole therapy
3.1±1.95 1.69±0.84 0.09±0.2**
5.84±2.68 3.02±1.53 0.2±0.25**
2.96±1.09 1.53±1.05 0.11±0.12*
Cumulative steroid dose (mg) at start of levamisole therapy after levamisole therapy
7704.7±3707.9
1316.0±1860.7**
7165.5±3115.2
1639.2±1689.1**
8086.5±4256.5
1505.0±1749.7** FRNS frequently relapsing nephrotic syndrome; SDNS steroid dependent nephrotic syndrome; SRNS steroid resistant nephrotic syndrome.
* P<0.01, ** P<0.0001 vs. start of levamisole therapy; data are given in mean±SD.
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Table 5 Relapses during levamisole therapy and during the follow-up period Number of relapsed
patients during levamisole therapy
Number of relapsed patients 24 month after levamisole therapy
FRNS (n=15) 2 3 SDNS (n=13) 6 2 SRNS (n=6) 3 1
FRNS frequently relapsing nephrotic syndrome; SDNS steroid dependent nephrotic syndrome; SRNS steroid resistant nephrotic syndrome.
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Table 6 Histopathology and therapeutic response
During levamisole therapy
No relapse 1 relapse 2 relapses
Histology
in 12 month
24 month after levamisole
therapy (number of relapses)
MCNS (n=13) 8 4 1 1 IgM NP (n=7) 3 4 - 1 FSGS (n=3) 2 1 - 1 Unknown histology (n=11) 10 1 - 3
MCNS minimal change nephrotic syndrome; IgM NP IgM nephropathy; FSGS focal segmental glomerulosclerosis.
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The endogenous creatinine clearance did not change significantly during the follow-
up period, being 108.2±45 ml/min per 1.73 m2 before the start of levamisole therapy and
108.0±47.7 ml/min per 1.73 m2 after its completion. Significant changes were not observed
in serum electrolyte and liver function parameters.
During the follow-up period, 5 of our patients developed reversible neutropenia
(white blood cell count <3x109/l). After normalization of the white blood cell count, the
levamisole treatment was restarted. None of our patients developed gastrointestinal,
cutaneous, or other side effects [20, 33−37]. Side effects of glucocorticoid therapy
disappeared during the observation period in all patients.
4.2. Results of the CP treated group
Table 7 and 8 present data on the treatment of patients according to histology and
clinical course of the disease. Twenty-two INS patients received CP for 2.5±0.5 months in
the first course as a second immunosuppressive drug after a remission with steroids was
achieved. The average time from the onset of NS to the start of CP therapy was 2.5±2.0
years. Fifteen of the 22 patients also received methylprednisolone (MP) pulse therapy before
administration of CP. Fifteen children had SDNS and seven had SRNS. Nineteen had MCNS
and three had FSGS. Proteinuria decreased from 3.9±2.9 to 0.5±1.4 g/day (p<0.01) and
remained at 0.9±1.7 g/day, 5 years later. One patient with FSGS, who had heavy proteinuria
(12.8 g/day) at the start of CP, still had 6.2 g/day at the end of the 5-year follow-up. There
were three patients with MCNS who developed increasing proteinuria, 1.8, 5.2 and 3.0 g/day,
respectively, by this time. Nine patients had a reduced creatinine clearance and were ranged
into National Kidney Foundation Kidney Disease Quality Outcomes Initiative [38] stages for
chronic kidney disease (NKF K/DOQI CKD) stage III-V at the end of CP therapy; one
normalized by the time of the 5-year follow-up. Eight of 22 patients had an elevated systolic
blood pressure (≥120 mmHg) at the end of the 5-year follow-up period.
23
Table 7 Statistical data of the cyclophosphamide and cyclosporine-A treated patients according to the clinical course (SDNS or SRNS) Group I (CP) Group II (CSA) Clinical classification SDNS (n=15) SRNS (n=7) SDNS (n=8) SRNS (n=7) Age at onset (year)
- mean±SD - median (range)
7.7±3.8 9 (2-14)
6.4±2.9
7.7 (2.2-12)
9.5±4.7
8 (4.5-16)
14.3±2.4
15 (10-16) Gender: male/female 12/3 3/4 7/1 ¾ Histology: MCNS/FSGS 12/3 7/0 4/4 4/3 Duration of treatment (month) (mean±SD)
2.5±0.5 2.4±0.5 20.2±8.5 36.7±17.2
Before 2 years after
5 years after
before 2 years after
5 years after
Before 2 years after
5 years after
before 2 years after
5 years after
Therapy
Urinary protein:creatinine ratio
- < 0.2 mg/mg
- 0.2-2.0 mg/mg - > 2.0 mg/mg
1 3 10
10 3 1
10 3 1
0 2 6
5 2 1
3 3 2
7 1 0
6 2 0
4 3 1
7 0 0
3 3 1
4 3 0
Urinary protein (g/24 h; mean±SD)
4.1±3.3
0.4±0.8∗
0.7±1.6∗
3.3±2.3
0.3±0.6∗
1.3±2∗
4.4±2.6
1.3±3.3∗
0.9±2∗
3.5±2.1
1.4±2.1∗
0.3±0.2∗
eGFR (ml/min per 1.73 m2), (NKF K/DOQI CKD stages)
- > 90 (stage I) - 60-89 (stage II)
- 30-59 (stage III) - 15-29 (stage IV) - < 15 (stage V)
4 5 4 1 1
5 4 3 3 0
7 3 3 1 1
1 5 0 0 1
1 3 2 1 0
4 0 1 1 1
2 4 1 1 0
0 4 3 0 1
5 1 1 0 1
0 3 2 0 1
2 2 2 1 0
3 3 0 1 0
systolic diastolic systolic diastolic systolic Diastolic systolic Diastolic BP (mmHg; mean±SD) - before th. - 1 year after th. - 2 years after th.
- 5 years after th.
108.3±12.3 111.2±12.3 113± 15.3 113.3±11.4
72±7 71.3±8.5 72.2±10.9 74.3±8.0
106.4±12.5 107.1±10.7 104.3±12.4 122.1±13.5
67.1±7.6 67.9±6.3 68.6±10.7 80±12.6
115.6±9.2 117.5±12.8 115±17.7
121.9±11.6
77.5±8.5 76.8±7.0 72.5±10 78.7±6.4
119.3±6 120.7±7.9 119.2±16.9 122.1±12.2
77.1±9 78.6±7.5 75±10.4
79.3±10.2 Relapse free period(month, mean±SD)
29.9±21.5 30.3±23.3 28.1±22.4 24±12.7
Relapse rate/year 5 years after therapy (mean±SD)
0±0.3∗
0.1±0.13∗
0.8±1.0∗
0.5±0.7∗
CP cyclophosphamide; CSA cyclosporine-A; SDNS steroid dependent nephrotic syndrome; SRNS steroid resistant nephrotic syndrome; MCNS minimal change nephrotic syndrome; FSGS focal segmental glomerulosclerosis; BP blood pressure; eGFR estimated glomerular filtration rate (according to the Schwarz formule); NKF K/DOQI CKD stages National Kidney Foundation K/DOQI stages for chronic kidney disease. ∗P<0.001 vs. before the start of CP or CSA therapy.
24
Table 8 Data of cyclophosphamide and cyclosporine-A treatment in idiopathic nephrotic syndrome according to the histology
Before starting therapy At the end of therapy Two years after treatment Five years after treatment
UP g/day
Cl creat ml/min per
1.73m2
Relapse rate/year
UP g/day
Cl creat ml/min
per 1.73m2
Relapse rate/year
UP g/day
Cl creat ml/min
per 1.73m2
Relapse rate/year
UP g/day
Cl creat ml/min per
1.73m2
Relapse rate/year
MCNS (n=19)
3.4±2.3 104±51.8 0.2±0.4∗ 88.4±28.2 0.2±0.4∗ 83.9±29 0.7±1.3∗ 108.2±45
FSGS (n=3)
6.6±5.4 77.5±34.3
2.5±3.5∗ 70.3±37.6
1.6±1.1∗ 75.6±29.5
2.1±3.4∗ 86.1±66.4
CP (n=22)
3.9±2.9 100.4±50 3.4±2.8 0.5±1.4∗ 85.9±29.3 0.5±0.6∗ 0.4±0.7∗ 82.7±28.5 0.2±0.1∗ 0.9±1.7∗ 105.3±47.7 0.1±0.2∗ MCNS (n=8)
3.5±2.3 83.8±27 0.7±1.1∗ 71±30.9 0.8±1.7∗ 86.9±22.6 0.2±0.2 ∗
80.7±52.5
FSGS (n=7)
4.5±2.4 88.6±29.2
1±1.7 ∗ 95.3±37.2
2.1±3.6∗ 80.2±26.7
1±2.2∗ 122.9±18.5
CSA (n=15)
3.9±2.3 86.6±27.3 3.7±3.1 0.9±1.4 83.9±35.4 0.9±0.5∗ 1.5±2.8 83.4±24.2 0.9±0.5∗ 0.7±1.6 109.8±35.5 0.6±0.8∗
CP cyclophosphamide; CSA cyclosporine-A; UP urinary protein (g/day); MCNS minimal change nephrotic syndrome; FSGS focal segmental glomerular sclerosis; Cl creat endogenous creatinine clearance (ml/min per 1.73 m2). Data are presented in mean±SD. ∗P<0.001 vs. before the start of CP or CSA therapy.
25
The relapse-free period was 30±21.5 months (29.9±21.5 months in SDNS and
30.3±23.3 months in SRNS patients). The relapse rates decreased significantly from 3.4±2.8
to 0.5±0.6/year during the first course of CP therapy and remained at this level (0.1±0.2/year)
during the 5-year follow-up (p<0.001). At the end of follow-up, five patients were in
remission (<0.4 g/day proteinuria, ≥80ml/min per 1.73 m2 creatinine clearance) with
immunosuppressive therapy.
In relation to CP therapy, 3/22 children had nausea, 2/22 patients had reversible hair
loss, 1/22 had leucopenia and 1/22 alopecia.
4.3. Results of the CSA treated group
Table 7 and 8 presents treatment data on the patients according to histology and
clinical course of the disease. Fifteen patients received CSA for an average of 28±15 months
in the first course as a second immunosuppressive drug after a remission with steroids had
been achieved. Twelve of the 15 patients also received MP pulse therapy before
administration of CSA. Time from diagnosis to the start of CSA treatment was 3.5±3 years.
Eight children had SDNS and seven SRNS. Seven of them had FSGS and eight MCNS.
Proteinuria decreased significantly (at the start of CSA treatment, it was 3.9±2.3 g/day,
whereas at the end of CSA treatment, it was 0.9±1.4 g/day, p<0.01, 5 years later, it was
0.7±1.6 g/day). Creatinine clearance and eGFR did not change significantly during the 2-year
follow-up but increased at the end of the study. Two patients with FSGS and one with MCNS
did not respond to this CSA course: proteinuria was 3.5; 2.5 and 4.4 g/day, respectively.
Proteinuria increased further in an FSGS case, to 6.1 g/day 5 years after the first CSA period,
whereas in the other FSGS and the MCNS patient, a further course of CSA treatment resulted
in a decrease in proteinuria to 0.4 and 0.5 g/day, respectively. After 5-years of follow-up,
eGFR decreased <15 ml/min per 1.73m2 in two FSGS patients. In these patients, marked
hypertension had also developed.
The relapse-free period was 26.2±18 months (28.1±22.4 months in SDNS and
24±12.7 months in SRNS patients). Relapse rates significantly decreased, from 3.7±3.1 to
0.9±0.5/year during the first CSA course and remained at this lower level (0.6±0.8/year)
26
during the follow-up (p<0.001). At the end of follow-up in the CSA group, four patients were
in remission (<0.4 g/day proteinuria, ≥80ml/min per 1.73 m2 creatinine clearance) with
immunosuppressant therapy.
During CSA treatment 3/15 patients exhibited hirsutism, 2/15 tremor, 2/15 gingival
hyperplasia, and 1/15 had nausea and appetite loss.
Table 9 represents the further therapy after the examined period. In the CP group,
CSA was introduced when CP results were not sufficient (6/22). Four of 22 patients received
prednisolone/MMF therapy as immunosuppression after CP. In the CSA group 5/15 patients
received more than one CSA course and 4/15 needed other immunosuppressive therapy. One
patient in each group needed renal replacement therapy at the end of the 5-year follow-up.
Figure 1 shows the clinical course according to the histology of the patients in
remission or in relapse during the follow-up period.
27
Table 9 Further immunosupressive treatment after first CP and CSA course Number
of patients
Number of patients
Only 1 course of CP 10 Only 1 course of CSA 5 CP after CP 0 CSA after CSA 5 Prednisolone and CSA after CP 2 Prednisolone and CSA after CSA 1 CSA after CP 4 CP after CSA 1 Prednisolone after CP 2 Prednisolone after CSA 1 MMF after CP 1 MMF after CSA 1 MMF and Prednisolone after CP 1 MMF and Prednisolone after CSA 0 HD/CAPD and CSA after CP 1 HD after CSA 1 CP cyclophosphamide; CSA cyclosporine-A; MMF mycophenolate mofetil; HD hemodialysis; CAPD continous ambulatory peritoneal dialysis.
28
Figure 1 Clinical course of CP and CSA treated patients after five years follow-up
SDNS (n=23) SRNS (n=14)
Group I (CP, n=15) Group II (CSA, n=8) Group I (CP, n=7) Group II (CSA, n=7)
remission, n=8 relapsed, n=6 RRT remission relapsed n (MCNS)=7 n (MCNS)=5 n (FSGS)=1 n (MCNS)=2 n (MCNS)=5 n (FSGS)=1 n (FSGS)=1 remission, n=3 relapsed, n=4 RRT remission relapsed, n=5 n (MCNS)=2 n (MCNS)=2 n (FSGS)=1 n (MCNS)=2 n (MCNS)=2 n (FSGS)=1 n (FSGS)=2 n (FSGS)=3 CP cyclophosphamide; CSA cyclosporine-A; SDNS steroid dependent nephrotic syndrome; SRNS steroid resistant nephrotic syndrome; RRT renal replacement therapy; MCNS minimal change nephrotic syndrome; FSGS focal segmental glomerular sclerosis.
29
5. DISCUSSION
5.1. Discussion of the levamisole study
One of the most difficult tasks in paediatric nephrology is the care of INS patients
with multiple relapses, and the situation is even more difficult in SDNS and SRNS patients.
These patients are candidates for treatment with steroid-sparing agents [15, 25, 19, 39].
Attempts to maintain remission and to prevent relapses are associated with certain
risks. The immunomodulatory agent levamisole has been used as an adjunctive therapy in
such patients [40−47] as a good alternative to major immunosuppressives [48]. This
treatment leads to decreases in the number of relapses and the amount of prednisolone
required. Niaudet et al. treated 30 SDNS patients with levamisole for a mean duration of 9.9
months. Almost half of the patients had no relapses, despite the achievement of a significant
reduction in the dose of prednisolone [41].
The British Association for Paediatric Nephrology (BAPN) reported prolonged
remission in patients with SDNS given alternate-day levamisole for 16 weeks [45]. However,
relapses occurred in the majority of patients after the treatment was stopped. Neuhaus at al.
likewise observed that levamisole at a dose of 2.5 mg/kg on alternate days for 6-18 months
was effective in inducing remission in more than half of the patients [15]. Bagga et al.
suggested that long-term levamisole therapy with low-dose prednisolone can lead to a
significant reduction in the relapse rate in SDNS patients [13]. In a prospective trial, Donia et
al. treated 20 SDNS patients with levamisole for 6 months and followed them for a further 6
months. At the end of the 6-month treatment period, 10 patients were maintaining remission
on levamisole alone. At the end of the 12-month study period, 5 patients (25 %) were still in
remission [47]. Ksiazek and Krynski reported a higher proportion (45.5 %) of patients who
were able to withdraw from steroids and maintain remission for more than 6 months after
levamisole therapy, perhaps because they did not stop levamisole throughout the study [49].
Tenbrock et al. similarly reported a 10-month duration of remission; they may have studied
FRNS patients [50]. Alsaran et al. concluded that levamisole is a good alternative to CP as a
second-line agent for FRNS patients [23]. Abeyagunawardena et al. reported a retrospective
30
comparative study in which levamisole was prescribed as a first steroid-sparing agent for 65
children; disease control was achieved in 30%. They concluded that levamisole is an
attractive steroid-sparing agent [18]. Boyer et al examined both short- and long-term efficacy
of levamisole (i.e. relapse frequency, cumulative annual steroid burden, somatic indices) in
10 children with SDNS. They gave levamisole for 12 months, and followed the patients for
three years (from one year prior levamisole treatment to the end of the 12th month after
cessation of the drug). They concluded that levamisole is an effective short- and long-term
steroid sparing agent [51].
We used approximately double the dose that was used in the BAPN study, because
they reported a number of relapses after the treatment was stopped. Therefore we decided to
use a higher dose. In our study, no relapse occurred in 23 of the 34 patients (FRNS, SDNS or
SRNS) during levamisole treatment, i.e. 67.6% remained in remission, while 10 patients had
one and one had two relapses in 12 months. In the 24-month follow-up period after
discontinuation of levamisole, 28 children remained in total remission and only 6 had
relapses, i.e. a remission rate of 82.3%. There were two relapses among the 15 FRNS
patients, six among the 13 SDNS children, and three among the 6 SRNS patients. Although
the number of our patients is small, the proportion of relapse-free patients during levamisole
treatment is 50% or higher in all three groups. These results are better than the previously
reported studies. At the same time one should be aware that this is a retrospective, non-
randomized study and there was no control group.
The small number of patients involved in the study does not allow a reliable statistical
analysis of the histological findings and the therapeutic effects during levamisole therapy.
Renal biopsy was performed in only 23 of 34 patients. It is therefore difficult to draw any
conclusion based on the total population when the histopathology was known in only about
75%. Nevertheless, our MCNS patients and those children with unknown histology
(indicating a clinical presentation that corresponds to MCNS) exhibited a better response to
levamisole than those with IgM NP and FSGS (Table 6). Genetic analysis of the patients was
not available in our centre at the time of the study.
The cumulative steroid dose was significantly lower following the introduction of
levamisole treatment (i.e. 1468±1766 mg/year). The positive effect of reducing the steroid
dosage may be secondary to the previously given alkylating agents. To investigate this, a
31
prospective study is necessary either with the elimination of alkylating agents or with a
separate group of patients treated with alkylating agents only versus alkylating agents
followed by levamisole.
The occurrence of side effects during levamisole treatment was rare. We found
reversible neutropenia in five patients, but no other side effects.
Some studies report, that other drugs may also have an important role in the treatment
of the examined population. High-dose mizoribine [52] or MMF [53] also appears to be
useful in maintaining remission and have a steroid-sparing effect in SDNS and SRNS
patients. They might be also successfully used in the CSA-dependent FRNS cases [52, 53].
5.2. Discussion of the CP versus CSA study
Our retrospective study documents that the long-term results of CP treatment in
childhood SDNS and SRNS are at least as effective as those for CSA.
5.2.1. Clinical course of nephrotic syndrome
In our study, CP treatment duration was 2.5±0.5 months, and the relapse-free period
was 30.05±21.5 months (in 5/22 patients >60 months). Eighteen of the 22 patients (82%) had
a relapse-free period of more than a year following CP therapy without other
immunosuppressive treatment. Altogether, 10/22 patients (48%) did not need further
immunosuppressive treatment until the end of the fifth year. Of the CP-treated patients,
45.5% were in complete and 36.3% in partial remission after the 5-year follow-up (81.8%
altogether). Further immunosuppressive therapy was needed in 12/22 patients because of
relapses.
In our CSA-treated group, CSA treatment duration was 28±15 months (7-60 months).
The relapse-free period following the start of CSA therapy was 26.2±18 months (in 2/15
patients it was >60 months). Of the CSA-treated patients, 46.6 % were in complete and
46.6% in partial remission (altogether 93.2%) at the end of the 5-year follow-up, and 33%
32
(5/15 patients) did not need therapy. Since many patients relapsed, their treatment was
resumed or changed (see table 9). Nevertheless, 9/15 patients still needed maintenance
immunosuppressant therapy from the beginning until the end of the 5-year-long follow-up.
At the end of the fifth year, 10/15 patients were in remission (66.6%) in the CSA
group and 15/22 (66.2%) in the CP group. The difference between the relapse-free period and
the relapse rates after the first CP or CSA period was significant after the 2-year and 5-year
follow-up period, with much rarer relapses following CP therapy (p<0.05). When estimating
our results, we found that partial remission is not equal complete remission and might finally
not turn out to be beneficial for the patients.
5.2.2. Renal function, proteinuria and hypertension
Compared with the CSA group, a significant decrease in creatinine clearance was
seen in patients who were treated with CP first after steroids, at the end of CP treatment, and
2 years after the CP therapy, but this change was reversible and disappeared after 5 years.
Endogenous creatinine clearance and eGFR also showed a significant increase in the CSA-
treated group after the 5 years of follow-up. In the CP-treated group, a significantly higher
proportion of patients had <0.4 g/day proteinuria (16/22 patients, 72.7%) than those in CSA
therapy (9/15 patients, 60%) at the end of the 5-year-long follow-up. At this time, blood
pressure was significantly lower after CP than following CSA therapy.
5.2.3. Renal histology
FSGS is an indicator for poor prognosis: 6/10 patients relapsed, 2/10 patients
developed end-stage renal failure and needed renal replacement therapy (RRT), and only
1/10 patient was in remission at the end of the fifth year with FSGS histology. In FSGS, the
likelihood of remission is much lower than in MCNS which should make a mixture of these
two groups obsolete. The low number of the FSGS patients in the CP-treated group (3/25)
compared to the 7/15 patients in the CSA group makes it difficult to determine the statistical
33
significance of our results. Furthermore, we cannot be sure about the homogeneity of these
two patient groups due to the lack of genetic analysis reviling the exact source of their
symptoms. Although our patients had documented steroid sensitivity at the onset,
controversially the histology and the refractory course ending in end-stage renal disease in
some patients suggests that some of them might have had a resistant or very poor response to
steroids initially.
5.2.4. Adverse events
The types of side-effects during CP and CSA therapy were different. CP therapy
applied for less than 3 months was not associated with more side-effects than CSA treatment.
Any complications in the two treatment groups proved to be reversible, though none of our
patients received more than one course of CP therapy. There were no patients who needed
hospitalization related to severe infections during the administration of oral CP or CSA.
The known long-term side-effects of CSA should be taken into account in the
treatment decision. Its use for more than 1 year can result in chronic nephrotoxicity in 17-
60% of patients [29] (i.e. CSA-associated arteriolopathy, tubulointerstitial lesions and focal
glomerular lesions [54]). Other side-effects include hypertension, gingival hyperplasia, and
hirsutism. Lijima et al. reported that CSA treatment duration and duration of heavy
proteinuria during CSA treatment were independent risk factors for the development of CSA-
induced tubulointerstitial lesions in children with MCNS who had been treated with long-
term moderate-dose CSA [55]. Also, when using CP, the physician should also take into
consideration the potential risk of infertility, cardiac disease and late cancer [56].
5.2.5. The possible use of cyclophosphamide or cyclosporine-A for treatment
Although a correct evaluation of the relative effectiveness of CP and CSA has not
been possible because of the lack of a head-to-head randomised trial, studies in which
alkylating agents and CSA have been compared have led to the conclusion that a course of
34
cytotoxic drug leads to a higher rate of cumulative sustained remission compared with CSA
[57, 58]. Pena et al. achieved high remission rates (73.3%) in histologically proven MCNS
and FSGS SRNS patients using MP pulses and alkylating agents (CP, Chl) together. They
also concluded that initial steroid resistance is a poor prognostic factor compared with late-
onset steroid resistance [59]. Our patients were steroid sensitive at the beginning, which may
explain our better results.
The efficiency of CP and Chl in patients with SDNS, SRNS, and FRNS is defined by
remission duration. The effect of cytotoxic drug therapy in SSNS depends on several factors,
such as the underlying glomerular disorder, e.g. MCNS, mild mesangial proliferation, IgM
NP [55, 60−62], FSGS, steroid sensitivity, FRNS or SDNS course of NS [63], type of
cytotoxic drug [64], drug dose [56, 65], treatment duration [66], and concomitant drug
therapy [60].
The reported remission rates after cytotoxic therapy vary from 0% after 12 months to
about 30% after 5 years [67, 68]. In approximately 10% of the patients, however, NS may
relapse during the cytotoxic treatment. The applied CP dose varies from 105 to 588 mg/kg
body weight cumulatively [69−71]. Higher dosages are associated with a higher chance of
relapse–free intervals, although the risk of side-effects increases. According to the meta-
analysis of cytotoxic treatment in FRNS children published by Latta et al., the remission
lasted for a maximum of 57 months after the first course of cytotoxic therapy, and the overall
relapse-free survival after 4 years is <50%. On average, studies on FRNS result in remission
rates of 72% after 2 years and 36% after 5 years; rates for SDNS are 40% and 24%,
respectively [33]. Second courses of cytotoxic therapy are reported to increase the rate of
long-lasting remissions [72−74]. We gave a CP dose of 112-168 mg/kg cumulatively in one
course, and we did not apply a second CP course in any of the NS patients. Our better results
may partially be explained by the fact that our patients had SSNS at the start of the disease.
In the study by Cattran et al., 49 SRNS due to FSGS patients were treated with CSA +
low-dose prednisolone and compared with low-dose prednisolone + placebo and followed for
200 weeks. They found 70% of the treatment group vs. 4% of the placebo group had a partial
or complete remission by 26 weeks. Relapse occurred in 40% of patients by week 52 and in
60% by week 78, but the remainder stayed in remission to the end of 200 weeks. There was a
35
decrease of 50% in the baseline creatinine clearance in 25% of the treated group compared
with 52% of the controls [75].
Ehrich et al. examined 86 children with SRNS due to FSGS and MCNS in a
retrospective, non-randomized study, and they concluded that prolonged and intensified
treatment with combined prednisolone + CSA therapy including intravenous
methylprednisolone pulses resulted in higher rate of remission when compared to CSA
monotherapy or other immunosuppressive therapies. Because of a lack of efficacy and the
risk of severe side effects of cytotoxic drugs, including gonadal toxicity, their experience
does not indicate that children should be treated for steroid-resistant MCNS with CP [76].
Planck et al. conducted a controlled multicentre randomized open label trial to test the
efficacy and safety of CSA (n=15) versus CP (n=17) pulses in the initial therapy of children
with newly diagnosed primary SRNS and histologically proven MCNS, FSGS or mesangial
hypercellularity. At week 12, nine of the 15 (60%) CSA patients showed at least partial
remission. In contrast, three of the 17 (17%) CP patients responded (p < 0.05). After 24
weeks, complete remission was reached by two of the 15 (13%) CSA and one of the 17 (5%)
CP patients (p = n.s.). Partial remission was achieved by seven of the 15 (46%) CSA and two
of the 15 (11%) CP patients (p<0.05). They concluded that CSA is more effective than CP in
inducing at least partial remission in SRNS in children [77]. Our better results can be
explained by the fact that our patients were steroid sensitive at the onset of their disease.
Based on the data of the above mentioned trial, Hodson et al. concluded that although
there was no difference in the number of patients achieving complete remission, those
patients receiving CSA treatment were significantly more likely to achieve partial remission
than those receiving intravenous CP. They also suggest that CSA rather than intravenous CP
should be used as first line therapy for children with SRNS [78].
In the Cochrane Review, 26 trials involving 1173 children were identified. CP and
Chl significantly reduced the relapse risk at 6-12 months compared with prednisolone alone.
In the single Chl vs. CP trial, no difference in relapse risk was observed at 2 years. There was
no difference at one year between intravenous and oral CP. CSA was as effective as CP and
Chl, and levamisole was more effective than steroids alone, but the effects were not
sustained, once CSA treatment was stopped. There was no difference in the risk for relapse
between MMF and CSA. Mizoribine and azathioprine were no more effective than placebo
36
or prednisone alone in maintaining remission. The reviewers concluded that 8-week-long
courses of CP or Chl and prolonged courses of CSA and levamisole reduce the risk of relapse
in children with relapsing SSNS compared with corticosteroids alone [5]. Meanwhile the
choice of agent depends on the physician and patient preferences related to therapy duration
and the type and frequency of complications [5].
Kemper et al. reported that SDNS can recur in patients despite CSA maintenance
therapy [79], and despite its efficacy, the majority of patients relapse if CSA is stopped [54,
80−82].
The role of the recently used drugs, such as tacrolimus, rituximab or the most hopeful
MMF was not examined in this study. Other studies report that the replacement of CSA with
the other calcineurin inhibitor tacrolimus did not lead to better management of severe SDNS
[83, 84].
37
6. CONCLUSIONS
There is no uniformed consensus as to the treatment of steroid–unresponsive NS.
Advances in pathogenesis, genetics and biomarkers or surrogate markers might be useful for
the diagnosis and identification of patients with steroid-unresponsive NS, severity of disease,
progression and response to therapy.
Our findings suggest that
1. levamisole significantly reduces the relapse rate and the cumulative steroid dose in
children with FRNS and SDNS and is also a beneficial and safe therapy for SRNS patients.
2. Levamisole may have a place in preventing relapse even after courses of akylating
agents, and it may also have some benefit for the treatment of SRNS patients.
The confirmation of the favourable effects of levamisole on the reduction of the
frequency of relapses and on sparing steroids in an adequately powered, double-blind,
placebo-controlled, randomized, multicenter clinical trial will promote consensus on the
place of levamisole in the treatment of FRNS in childhood.
3. In the long term, both CP and CSA are effective second-line therapies following
steroid monotherapy in INS patients, but
4. the relapse rate is lower and the relapse-free period significantly longer in the CP-
treated group.
5. An important message of our study is that most children who have a difficult course
of NS after an initial remission induced by steroids do well 7-8 years after presentation.
6. A good remission rate can be achieved after 5 years following the initial CP and CSA
therapy, and the incidence of side effects is low.
On the other hand, our study is not able to give a final an answer to the question as to
which is the better treatment in the examined patient population due to its retrospective
nature with unequal distribution of FSGS in the different treatment groups. In addition, no
supplementary genetic studies were performed; therefore about 10% of our patients are
expected to have a genetic origin behind of their symptoms.
Further research is needed to elucidate the disorder’s molecular pathogenesis, identify
new prognostic indicators and to develop better approaches to treatment.
38
ACKNOWLEDGEMENTS
I would like to express my sincere thanks to Prof. Sándor Túri, M.D., Ph.D., D.Sc.,
Director of the Department of Paediatrics at the University of Szeged, for his invaluable
help, support and encouragement during my clinical and research activities, as well as for his
scientific supervision in the Doctoral School of Clinical Medicine of the University of
Szeged.
I appreciate the support and help of my colleagues, Ibolya Haszon, M.D., Ph.D.,
Csaba Bereczki M.D., Ph.D., and Ferenc Papp M.D., Nephrology Division of Department of
Paediatrics, University Szeged, who also involved me, as a team member, in their research
activities.
I am grateful to Gyula Tálosi M.D. PhD., Department of Paediatrics, University
Szeged, for the survey and the suggestions he made.
I would like to thank Máté Sümegi Ph.D., Institute of Experimental Medicine,
Hungarian Academy of Sciences, for his technical support and for the language review of
this PhD Thesis.
39
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